Functional aspects of 17beta-hydroxysteroid dehydrogenase 1 determined by comparison to a closely related retinol dehydrogenase

Determining the functional aspects of a gene or protein is a difficult and time-consuming process. De novo analysis is surely the hardest and so it is often quite useful to start with a comparison to functionally or structurally related proteins. Although 17beta-hydroxysteroid dehydrogenase type 1 (17beta-HSD 1) can hardly be called a new protein but rather the best characterized among the family of 17beta-HSDs some aspects of structure–function relationships remain unclear. We have sought new aspects of 17beta-HSD 1 function through a comparison with its closest homolog, a photoreceptor-associated retinol dehydrogenase (prRDH). Overall amino acid identity and size of the proteins are highly conserved, but major differences occur in the C-termini, where prRDH, but not 17beta-HSD 1, harbors motifs indicative of membrane localization. To gain insight into substrate discrimination by prRDH and 17beta-HSD 1, we constructed 3D-structure models of the corresponding zebrafish enzymes. Investigation of the substrate binding site revealed a few identical amino acids, and suggested a role for G143 in zebrafish 17beta-HSD 1 and M146 and M147 in the two zebrafish paralogs prRDH 1 and prRDH 2, respectively, in substrate specificity. Activity measurements of modified proteins in transiently transfected intact HEK 293 cells hint at a putative role of these amino acids in discrimination between steroid and retinoid substrates.     

Short chain dehydrogenase/reductase rdhe2 is a novel retinol dehydrogenase essential for frog embryonic development

The enzymes responsible for the rate-limiting step in retinoic acid biosynthesis, the oxidation of retinol to retinaldehyde, during embryogenesis and in adulthood have not been fully defined. Here, we report that a novel member of the short chain dehydrogenase/reductase superfamily, frog sdr16c5, acts as a highly active retinol dehydrogenase (rdhe2) that promotes retinoic acid biosynthesis when expressed in mammalian cells. In vivo assays of rdhe2 function show that overexpression of rdhe2 in frog embryos leads to posteriorization and induction of defects resembling those caused by retinoic acid toxicity. Conversely, antisense morpholino-mediated knockdown of endogenous rdhe2 results in phenotypes consistent with retinoic acid deficiency, such as defects in anterior neural tube closure, microcephaly with small eye formation, disruption of somitogenesis, and curved body axis with bent tail. Higher doses of morpholino induce embryonic lethality. Analyses of retinoic acid levels using either endogenous retinoic acid-sensitive gene hoxd4 or retinoic acid reporter cell line both show that the levels of retinoic acid are significantly decreased in rdhe2 morphants. Taken together, these results provide strong evidence that Xenopus rdhe2 functions as a retinol dehydrogenase essential for frog embryonic development in vivo. Importantly, the retinol oxidizing activity of frog rdhe2 is conserved in its mouse homologs, suggesting that rdhe2-related enzymes may represent the previously unrecognized physiologically relevant retinol dehydrogenases that contribute to retinoic acid biosynthesis in higher vertebrates.     

Identification and characterization of retinoid-active short-chain dehydrogenases/reductases in Drosophila melanogaster

Background

In chordates, retinoid metabolism is an important target of short-chain dehydrogenases/reductases (SDRs). It is not known whether SDRs play a role in retinoid metabolism of protostomes, such as Drosophila melanogaster.

Methods

Drosophila genome was searched for genes encoding proteins with ∼ 50% identity to human retinol dehydrogenase 12 (RDH12). The corresponding proteins were expressed in Sf9 cells and biochemically characterized. Their phylogenetic relationships were analyzed using PHYLIP software.

Results

A total of six Drosophila SDR genes were identified. Five of these genes are clustered on chromosome 2 and one is located on chromosome X. The deduced proteins are 300 to 406 amino acids long and are associated with microsomal membranes. They recognize all-trans-retinaldehyde and all-trans-3-hydroxyretinaldehyde as substrates and prefer NADPH as a cofactor. Phylogenetically, Drosophila SDRs belong to the same branch of the SDR superfamily as human RDH12, indicating a common ancestry early in bilaterian evolution, before a protostome–deuterostome split.

Conclusions

Similarities in the substrate and cofactor specificities of Drosophila versus human SDRs suggest conservation of their function in retinoid metabolism throughout protostome and deuterostome phyla.

General significance

The discovery of Drosophila retinaldehyde reductases sheds new light on the conversion of β-carotene and zeaxantine to visual pigment and provides a better understanding of the evolutionary roots of retinoid-active SDRs.     

The crystal structure of 3alpha -hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni shows a novel oligomerization pattern within the short chain dehydrogenase/reductase family

The crystal structure of 3alpha-hydroxysteroid dehydrogenase/carbonyl reductase from Comamonas testosteroni (3alpha-HSDH) as well as the structure of its binary complex with NAD(+) have been solved at 1.68-A and 1.95-A resolution, respectively. The enzyme is a member of the short chain dehydrogenase/reductase (SDR) family. Accordingly, the active center and the conformation of the bound nucleotide cofactor closely resemble those of other SDRs. The crystal structure reveals one homodimer per asymmetric unit representing the physiologically active unity. Dimerization takes place via an interface essentially built-up by helix alphaG and strand betaG of each subunit. So far this type of intermolecular contact has exclusively been observed in homotetrameric SDRs but never in the structure of a homodimeric SDR. The formation of a tetramer is blocked in 3alpha-HSDH by the presence of a predominantly alpha-helical subdomain which is missing in all other SDRs of known structure.     

Analysis of the NADH-dependent retinaldehyde reductase activity of amphioxus retinol dehydrogenase enzymes enhances our understanding of the evolution of the retinol dehydrogenase family

In vertebrates, multiple microsomal retinol dehydrogenases are involved in reversible retinol/retinal interconversion, thereby controlling retinoid metabolism and retinoic acid availability. The physiologic functions of these enzymes are not, however, fully understood, as each vertebrate form has several, usually overlapping, biochemical roles. Within this context, amphioxus, a group of chordates that are simpler, at both the functional and genomic levels, than vertebrates, provides a suitable evolutionary model for comparative studies of retinol dehydrogenase enzymes. In a previous study, we identified two amphioxus enzymes, Branchiostoma floridae retinol dehydrogenase 1 and retinol dehydrogenase 2, both candidates to be the cephalochordate orthologs of the vertebrate retinol dehydrogenase enzymes. We have now proceeded to characterize these amphioxus enzymes. Kinetic studies have revealed that retinol dehydrogenase 1 and retinol dehydrogenase 2 are microsomal proteins that catalyze the reduction of all-trans-retinaldehyde using NADH as cofactor, a remarkable combination of substrate and cofactor preferences. Moreover, evolutionary analysis, including the amphioxus sequences, indicates that Rdh genes were extensively duplicated after cephalochordate divergence, leading to the gene cluster organization found in several mammalian species. Overall, our data provide an evolutionary reference with which to better understand the origin, activity and evolution of retinol dehydrogenase enzymes.     

Extended superfamily of short alcohol-polyol-sugar dehydrogenases: structural similarities between glucose and ribitol dehydrogenases

The recently determined primary structure of glucose dehydrogenase from Bacillus megaterium was scanned by computerized comparisons for similarities with known polyol and alcohol dehydrogenases. The results revealed a highly significant similarity between this glucose dehydrogenase and ribitol dehydrogenase from Klebsiella aerogenes. Sixty-one positions of the 262 in glucose dehydrogenase are identical between these two proteins (23% identity), fitting into a homology alignment for the complete polypeptide chains. The extent of similarity is equivalent to that between other highly divergent but clearly related dehydrogenases (two zinc-containing alcohol dehydrogenases, 25% sorbitol and zinc-containing alcohol dehydrogenases, 25%; ribitol and non-zinc-containing alcohol dehydrogenases, 20%), and suggests an ancestral relationship between glucose and ribitol dehydrogenases from different bactera. The similarities fit into a previously suggested evolutionary scheme comprising short and long alcohol and polyol dehydrogenases, and greatly extend the former group to one composed of non-zinc-containing alcohol-polyol-glucose dehydrogenases.     

Comparative genomic and phylogenetic analysis of short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity

Human short-chain dehydrogenases/reductases with dual retinol/sterol substrate specificity (RODH-like enzymes) are thought to contribute to the oxidation of retinol for retinoic acid biosynthesis and to the metabolism of androgenic and neuroactive 3alpha-hydroxysteroids. Here, we investigated the phylogeny and orthology of these proteins to understand better their origins and physiological roles. Phylogenetic and genomic analysis showed that two proteins (11-cis-RDH and RDHL) are highly conserved, and their orthologs can be identified in the lower taxa, such as amphibians and fish. Two other proteins (RODH-4 and 3alpha-HSD) are significantly less conserved. Orthologs for 3alpha-HSD are present in all mammals analyzed, whereas orthologs for RODH-4 can be identified in some mammalian species but not in others due to species-specific gene duplications. Understanding the evolution and divergence of RODH-like enzymes in various vertebrate species should facilitate further investigation of their in vivo functions using animal models.     

Ultrastructure of the distal retina of the adult zebrafish, Danio rerio

The organization, morphological characteristics, and synaptic structure of photoreceptors in the adult zebrafish retina were studied using light and electron microscopy. Adult photoreceptors show a typical ordered tier arrangement with rods easily distinguished from cones based on outer segment (OS) morphology. Both rods and cones contain mitochondria within the inner segments (IS), including the large, electron-dense megamitochondria previously described (Kim et al.) Four major ultrastructural differences were observed between zebrafish rods and cones: (1) the membranes of cone lamellar disks showed a wider variety of relationships to the plasma membrane than those of rods, (2) cone pedicles typically had multiple synaptic ribbons, while rod spherules had 1-2 ribbons, (3) synaptic ribbons in rod spherules were ∼2 times longer than ribbons in cone pedicles, and (4) rod spherules had a more electron-dense cytoplasm than cone pedicles. Examination of photoreceptor terminals identified four synaptic relationships at cone pedicles: (1) invaginating contacts postsynaptic to cone ribbons forming dyad, triad, and quadrad synapses, (2) presumed gap junctions connecting adjacent postsynaptic processes invaginating into cone terminals, (3) basal junctions away from synaptic ribbons, and (4) gap junctions between adjacent photoreceptor terminals. More vitread and slightly farther removed from photoreceptor terminals, extracellular microtubule-like structures were identified in association with presumed horizontal cell processes in the OPL. These findings, the first to document the ultrastructure of the distal retina in adult zebrafish, indicate that zebrafish photoreceptors have many characteristics similar to other species, further supporting the use of zebrafish as a model for the vertebrate visual system.     

Exploring the active site of yeast xylose reductase by site-directed mutagenesis of sequence motifs characteristic of two dehydrogenase/reductase family types

Starting from a common tyrosine, yeast xylose reductases (XRs) contain two conserved sequence motifs corresponding to the catalytic signatures of single-domain reductases/epimerases/dehydrogenases (Tyrⁿ-(X)₃-Lysⁿ⁺⁴) and aldo/keto reductases (AKRs) (Tyrⁿ-(X)₂₈-Lysⁿ⁺²⁹). Tyr⁵¹, Lys⁵⁵ and Lys⁸⁰ of XR from Candida tenuis were replaced by site-directed mutagenesis. The purified Tyr⁵¹→ Phe and Lys⁸⁰→Ala mutants showed turnover numbers and catalytic efficiencies for NADH-dependent reduction of -xylose between 2500- and 5000-fold below wild-type levels, suggesting a catalytic role of both residues. Replacing Lys⁵⁵ by Asn, a substitution found in other AKRs, did not detectably affect binding of coenzymes, and enzymatic catalysis to carbonyl/alcohol interconversion. The contribution of Tyr⁵¹ to rate enhancement of aldehyde reduction conforms with expectations for the general acid catalyst of the enzymatic reaction.     

HPLC/UV quantitation of retinal, retinol, and retinyl esters in serum and tissues

We report robust HPLC/UV methods for quantifying retinyl esters (RE), retinol (ROL), and retinal (RAL) applicable to diverse biological samples with lower limits of detection of 0.7, 0.2, and 0.2 pmol, respectively, and linear ranges greater than 3 orders of magnitude. These assays function well with small, complex biological samples (10-20 mg tissue). Coefficients of variation range from 5.9 to 10.0% (intraday) and from 5.9 to 11.0% (interday). Quantification of endogenous RE, ROL, and RAL in mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, skin, brain, and brain regions) reveals utility. Ability to discriminate spatial concentrations of ROL and RE is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum). We also developed a method to distinguish isomeric forms of ROL to investigate precursors of retinoic acid. The ROL isomer assay has limits of detection between 3.5 and 4.5 pmol and has a linear range and coefficient of variation similar to those of the ROL/RE and RAL assays. The assays described here provide for sensitive and rigorous quantification of endogenous RE, ROL, and RAL to elucidate retinoid homeostasis in disease states such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.     

Characterization of a novel airway epithelial cell-specific short chain alcohol dehydrogenase/reductase gene whose expression is up-regulated by retinoids and is involved in the metabolism of retinol

Multiple retinoic acid responsive cDNAs were isolated from a high density cDNA microarray membrane, which was developed from a cDNA library of human tracheobronchial epithelial cells. Five selected cDNA clones encoded the sequence of the same novel gene. The predicted open reading frame of the novel gene encoded a protein of 319 amino acids. The deduced amino acid sequence contains four motifs that are conserved in the short-chain alcohol dehydrogenase/reductase (SDR) family of proteins. The novel gene shows the greatest homology to a group of dehydrogenases that can oxidize retinol (retinol dehydrogenases). The mRNA of the novel gene was found in trachea, colon, tongue, and esophagus. In situ hybridization of airway tissue sections demonstrated epithelial cell-specific gene expression, especially in the ciliated cell type. Both all-trans-retinoic acid and 9-cis-retinoic acid were able to elevate the expression of the novel gene in primary human tracheobronchial epithelial cells in vitro. This elevation coincided with an enhanced retinol metabolism in these cultures. COS cells transfected with an expression construct of the novel gene were also elevated in the metabolism of retinol. The results suggested that the novel gene represents a new member of the SDR family that may play a critical role in retinol metabolism in airway epithelia as well as in other epithelia of colon, tongue, and esophagus.     

Microtubules and 10 nm-filaments in the retinal pigment epithelium during the diurnal light-dark-cycle

Summary Microtubules and 10 nm-filaments appear to be involved in the functions of the retinal pigment epithelium (RPE). The presence of microtubules in the RPE of light-adapted eyes, but not in dark-adapted eyes, suggests that they may be involved primarily in organelle movement. On the other hand, the random and constant presence of 10 nm-filaments within the basal portion of the PE implies a cytoskeletal role for these filaments.The authors thank their colleagues Pierre Couillard and Michel Anctil for helpful advice and criticism during the course of this study. Financial support was provided by the C.R.S.N.G. du Canada and the Ministère de l'Education du Québec (F.C.A.C.)     

Evidence of membrane transformation during melanogenesis. Electron microscopic study on the retinal pigment epithelium of chick embryos

Summary Some characteristics of early premelanosomes (PM) suggest that primarily a continuous cisternal complex of the endoplasmic reticulum (ER) is transformed simultaneously to PM. These characteristics are: (i) the form and size, which are similar to ER cisternae; (ii) the localization in groups in the ER; (iii) the same stage of maturation within a group; (iv) the continuities between early PM, and (v) the lack of continuities between ER and PM. Comparative measurements reveal that the limiting membrane of PM, with a total thickness of 7.6±0.19 nm and a center-to-center distance of 5.2±0.06 nm, is significantly thicker than the ER membrane (6.3±0.15 nm and 4.3±0.04 nm, respectively) and the melanosome limiting membrane (6.5±0.22nm and 4.4±0.05 nm, respectively). Therefore, during the formation of melanosomes, the limiting membrane must be transformed from a thin (ER) to a thick (PM) and again to a thin (melanosome) state.     

Kinetic characterization of recombinant mouse retinal dehydrogenase types 3 and 4 for retinal substrates

Background

Retinal dehydrogenases (RALDHs) catalyze the dehydrogenation of retinal into retinoic acids (RAs), which are required for embryogenesis and tissue differentiation. This study sought to determine the detailed kinetic properties of 2 mouse RALDHs, namely RALDH3 and 4, for retinal isomer substrates, to better define their specificities in RA isomer synthesis.

Methods

RALDH3 and 4 were expressed in Escherichia coli as His-tagged proteins and affinity-purified. Enzyme kinetics were performed with retinal isomer substrates. The enzymatic products were analyzed by high pressure liquid chromatography.

Results

RALDH3 oxidized all-trans retinal with high catalytic efficiency (V_max/K_m = 77.9) but did not show activity for either 9-cis or 13-cis retinal substrates. On the other hand, RALDH4 was inactive for all-trans retinal substrate, exhibited high activity for 9-cis retinal oxidation (V_max/K_m = 27.4), and oxidized 13-cis retinal with lower catalytic efficiency (V_max/K_m = 8.24). β-ionone, a potent inhibitor of RALDH4 activity, suppressed 9-cis and 13-cis retinal oxidation competitively with inhibition constants of 0.60 and 0.32, respectively, but had no effect on RALDH3 activity. The divalent cation MgCl₂ activated 13-cis retinal oxidation by RALDH4 by 3-fold, did not significantly influence 9-cis retinal oxidation, and slightly activated RALDH3 activity.

Conclusions

These data extend the kinetic characterization of RALDH3 and 4, providing their specificities for retinal isomer substrates.

General significance

The kinetic characterization of RALDHs should give useful information in determining amino acid residues that are involved in the specificity for retinal isomers and on the role of these enzymes in the synthesis of RAs in specific tissues.     

苹果酸脱氢酶的结构及功能

苹果酸脱氢酶（MDH）可以催化苹果酸与草酰乙酸间的可逆转换,主要参与TCA循环、光合作用、C4循环等代谢途径。苹果酸脱氢酶可分为NAD-依赖性的MDFI（NAD—MDH）和NADP-依赖性的MDH（NADP—MDH）。在所有真核生物和大部分细菌中,MDH通常形成同源二聚体,在少数细菌中为四聚体。不同来源的MDH催化机制和它们的动力学性质十分类似,显示了它们具有高度的结构相似性。MDH的功能多样,包括线粒体中的能量提供和植物的活性氧代谢等。回顾了苹果酸脱氢酶在生理学、医学、农学领域的研究进展,并针对其生化特性、空间结构特点、催化机理等生物学功能的分子生物学进展进行了综述。     

Comparative transcript and alkaloid profiling in Papaver species identifies a short chain dehydrogenase/reductase involved in morphine biosynthesis

Plants of the order Ranunculales, especially members of the species Papaver, accumulate a large variety of benzylisoquinoline alkaloids with about 2500 structures, but only the opium poppy (Papaver somniferum) and Papaver setigerum are able to produce the analgesic and narcotic morphine and the antitussive codeine. In this study, we investigated the molecular basis for this exceptional biosynthetic capability by comparison of alkaloid profiles with gene expression profiles between 16 different Papaver species. Out of 2000 expressed sequence tags obtained from P. somniferum, 69 show increased expression in morphinan alkaloid-containing species. One of these cDNAs, exhibiting an expression pattern very similar to previously isolated cDNAs coding for enzymes in benzylisoquinoline biosynthesis, showed the highest amino acid identity to reductases in menthol biosynthesis. After overexpression, the protein encoded by this cDNA reduced the keto group of salutaridine yielding salutaridinol, an intermediate in morphine biosynthesis. The stereoisomer 7-epi-salutaridinol was not formed. Based on its similarities to a previously purified protein from P. somniferum with respect to the high substrate specificity, molecular mass and kinetic data, the recombinant protein was identified as salutaridine reductase (SalR; EC 1.1.1.248). Unlike codeinone reductase, an enzyme acting later in the pathway that catalyses the reduction of a keto group and which belongs to the family of the aldo-keto reductases, the cDNA identified in this study as SalR belongs to the family of short chain dehydrogenases/reductases and is related to reductases in monoterpene metabolism.